Ugur Deligezer, Ebru Esin Akisik, Nejat Dalay
A novel application of melting curves: utility of peak area calculation for relative methylation quantification
Background: DNA methylation markers appear to be useful in cancer detection, in evaluating the prognosis associated with disease progression, and in detecting the metastatic potential of tumors.
Methods: We present an approach for relative quantification of methylated gene sequences. The technique combines conventional PCR and LightCycler fluorescence PCR. In the conventional PCR step, bisulfite-modified molecules are selectively enriched, irrespective of their methylation status, and used as template in the second step, the LightCycler fluorescence PCR, which includes methylation-specific primers and fluorescently labeled probes. After amplification, a stepwise increase in temperature leads to the generation of melting curves reflecting fluorescence emission induced by disassociation of the probe-target complexes. Quantification is based on calculation of the peak areas for melting curves using a Gaussian fit curve. The area under this curve indicates the relative amounts of methylated sequences in each sample.
Results: By normalizing the peak areas by template concentrations, a methylation index was calculated for each sample. This approach was then used to carry out a relative quantification of p16INK4A gene methylation in bone marrow and blood mononuclear cells from patients with positive translocation status.
Conclusions: If validated in further studies, this approach represents a highly specific technique that can be used in analyzing paired samples of cancer patients.
Clin Chem Lab Med 2007;45:867–73.
Clinical Chemical Laboratory Medicine, Walter de Gruyter
Print ISSN: 1434-6621
Volume: 45, 07/2007
Pages: 867 - 873
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